The stem cell biologists describe the process of formation of blood as Haemopoiesis. This word is derived from the Greek root “haima” means blood and “poiesis” means to form. This process commences with the primitive haemopoietic stem cell. All blood cells originate from this cell. Stem cell transplantation has become an integral part of modern therapy. This regulated production of blood cells is one of the areas in biology most, amenable to scientific investigation. Blood cells, keratinocytes in skin & epithelium in gut have a short life span and are constantly renewed in specialized centres. The most discrete haemopoietic tissues are bone marrow, spleen, lymph nodes & thymus. Prenatally haemopoiesis also occurs in embryonic yolk sac from where it shifts to the liver and finally to the bone-marrow.
Haemopoiesis in mammals during embryonic & foetal development in humans is a migratory phenomenon. It begins in the foci of the yolk sac known as blood islands & migrates to the liver & spleen followed by bone marrow. The site of haemopoiesis varies with age. In the developing foetus, haemopoiesis commences in the yolk sac but after birth it is present solely in the bone marrow. In the infant, all the bone marrow space is filled with actively dividing haemopoietic cells and because of its red appearance (result of erythropoiesis) it is often called red marrow. This active haemopoiesis is necessary to meet the demands for blood cells by the growing child. It is noticed that with increase in age the amount of bone marrow space dedicated to haemopoiesis becomes restricted and active red marrow is replaced by fat cells and the marrow takes on a yellow appearance. The bone marrow is a richly cellular connective tissue within the bones of the body, specialized to produce blood cells & deliver them into circulation. It is the major haemopoietic tissue in human beings from the fifth week of foetal life through adulthood & accounts for about 5% of adult body weight. The cells of bone marrow consists of 1–2% of stem cells and the rest of differentiating and differentiated cells.
Haemopoietic stem cells are distinguished from other haemopoietic cells by their capacity to generate new stem cells, a process termed “self renewal” & their ability to proliferate & differentiate into different lineages thus clonally regenerating all the different types of blood cells. These processes of self renewal & differentiation are regulated by haemopoietic microenvironment. Haemopoietic stem cells comprise of a very small part of the haemopoietic system. These stem cells have an almost unlimited proliferative capacity which can be gauged by the fact that about 3.7×106 myeloid cells need to be produced every day of adult human life under normal conditions. During severe infections the demand for the differentiated end products of these cells may increase by 10 fold. Such a tremendous proliferative capacity needs very high regulation which is provided by the numerous cytokines acting in conjunction with each other & with other cell types regulating haemopoiesis through cell to cell contact. The bone marrow which is highly structured tissue is an ideal haven for this process to occur as it has the capacity to simultaneously provide different microenvironments to regulate the production of these cells as per the demands posed by the body.
As the bone marrow is the seat of haemopoiesis, transplantation of this marrow can be of therapeutic importance in various haematological diseases including malignancies. In recent years although significant advances have been made in bone marrow transplantion (BMT), the basic problem of finding a suitable matched donor still remains. This is because a group of antigens expressed by the leukocytes called human leukocyte antigens (HLA) need to match between the donor & the recipient. Also bone marrow harvesting is a painful & invasive procedure & it is difficult to get a suitable marrow donors as many donors are unwilling to donate their bone marrow. If bone marrow from a donor is used for a different person, the transplant is called an “allogeneic” BMT and when the bone marrow is used for an identical twin, the transplant is called “syngeneic” BMT. In an allogeneic BMT, it is essential that the new bone marrow infused into the patient matches with the genetic makeup of the patients own marrow as perfectly as possible. Special tests are conducted to determine whether or not the donor's bone marrow matches with the patient. The bone marrow transplant by conventional method requires the patient to be healthy enough to undergo the rigors of the transplant procedure. It requires an expert medical team who are well experienced in bone marrow transplant. It also requires providing patients and their families with emotional and psychological support before and after the transplant. The bone marrow is a debilitating experience. It can take as long as a year for the new bone marrow to function normally. Patients undergoing bone marrow transplant are closely monitored during this time to identify any infections or complications that can develop.
Looking to the problems associated with the bone marrow transplant stated above, alternative sources of stem cells are being investigated. The most important alternative source of haemopoietic stem cells is that derived from cord blood.
The cord blood as a source is advantageous to the traditional stem cell source i.e. the bone marrow, in the following ways:    1. The cord blood is much easier to obtain than bone marrow.    2. It can be collected immediately after birth of a child and does not interfere with normal delivery process.    3. The number of potential donors is high hence the question of getting a match for a patient does not arise.    4. The cord blood stem cells are naive and express HLA antigens poorly, especially the class II antigens, which are important in transplantation. As the antigens are poorly expressed, the two or three antigen mismatch does not result in graft versus host disease (GVHD) and the consequent rejection, thereby making the process of transplanting cord blood stem cells easier as compared to bone marrow transplantation.A major limitation of using cord blood for transplantation is that the yield of stem cells may not be enough for transplantation in adults. This can however be overcome by the expansion technique described for a quantitative increase in the stem cell of small volume collections of cord blood. The collected sample sometimes does not contain enough cells for complete haemopoietic reconstitution of an adult. Thus, for a cord blood to become a viable transplantable source of stem cells these cells must be increased by expansion in vitro, without the loss in their stemness. The ability of haemopoietic stem cells to express an intrinsic expansion & proliferation potential in vitro depends on the cytokines present in a culture. Several stem cell populations depending on their maturity undergo extensive proliferation & differentiation in response to stimulation with different combinations of cytokines. Cytokine mediated expansion therefore has been proposed as a means of increasing the total number of cells as well as committed & primitive haemopoietic progenitors in cord blood (About 1994). However, it is noticed that cytokines mediated expansion compromise the long term repopulating capacity of stem cells.
Different techniques are being used for the expansion of stem cells from umbilical cord blood. Many Biotech companies already own dozens of patents on the culture techniques of stem cells. However, they suffer from some defects. The stem cells that are cultured, tend to lose their stemness/self-renewal property.
Looking to the increased therapeutic potential for stem cell isolation, the inventors of the present invention have developed a device/bioreactor and a process for expansion of stem cells, for therapeutic use, from cord blood without compromising their self-renewal property. The processes of self-renewal and differentiation are regulated by haemopoietic growth factors and haemopoietic microenvironment, which the device of the present invention has regulated, in a manner that the expansion system used retains and expands stem cells with long term repopulating potential.
A bio-reactor is a device for growing cells. The bioreactor of the present invention has a scaffolding material placed on the base of the culture vessel. It is the scaffolding material that provides the microenvironment substantially identical to that present in the human bone marrow. The scaffolding material is porous in nature with sufficient internal space for the stem cells to grow. The device/bio-reactor of the present invention is easy to use, reproducible and particularly suitable for large scale cord blood processing under Good Manufacturing Practice (GMP) conditions.